武汉大学生命科学学院：《分子生物学》（英文版）How to Properly Use and Maintain Laboratory Equipment

輯 RAD/US (mm/ Figure 4.4 Deration Curve. Reproduced with permission of Kendro Laboratory manufacturers can provide the appropriate conditions for step runs on your rotor-centrifuge combination Troubleshooting How Can you best avoid service calls? Too often operating manuals are buried in unmarked drawers never to be seen again. This is a costly error because manuals contain information that can often solve problems without the expense and delay of a service call Older instruments may have brush motors. The more frequently Troutman et al

64 Troutman et al. manufacturers can provide the appropriate conditions for step￾runs on your rotor-centrifuge combination. Troubleshooting How Can You Best Avoid Service Calls? Too often operating manuals are buried in unmarked drawers, never to be seen again. This is a costly error because manuals contain information that can often solve problems without the expense and delay of a service call. Older instruments may have brush motors.The more frequently Figure 4.4 Deration Curve. Reproduced with permission of Kendro Laboratory Products

Figure 4.5 Operation of vertical rotors.(a) The gradi- centrifugal force ent is prepared, the sample is layered on top, and the cen- trifuge the pockets of the vertical rotor.(b) Both sample and gradient begin to reorient as rotor accelerates.(c)Reori. entation of the sample and gradient is now complete. (d) Bands form as the parti. cles sediment.(e) Bands and gradient reorient as the rotor decelerates. )Bands and gradient both fully rori- ented: rotor at rest. Fro Centifugation: A Practical Approach(2nd Ed. ) 1984 Rickwood, D, ed. Reprinted n of oxford uni. Figure 4.6 Effect of Triton X-100 on RNA pellet in the A concentration of Triton x 100 in these tubes is(a)0% (b)0.0001%,(c)0.001%,and (d)1%. As the Triton X-100 concentration increases, the pellet adhesion decreases. High Triton X-100 leve reddish band at the top of the ube. Courtesv. Beckman Coulter, Inc, Fullerton, CA D A-1790A. How to Properly Use and Maintain Laboratory Equipment

How to Properly Use and Maintain Laboratory Equipment 65 A B C D Figure 4.6 Effect of Triton X-100 on RNA pellet in the TLN-100 rotor. The final concentration of Triton X- 100 in these tubes is (a) 0%, (b) 0.0001%, (c) 0.001%, and (d) 1%. As the Triton X-100 concentration increases, the pellet adhesion decreases. High Triton X-100 levels (0.1–1%) produce a visible reddish band at the top of the tube. Courtesy, Beckman Coulter, Inc., Fullerton, CA, application note A-1790A. Figure 4.5 Operation of vertical rotors. (a) The gradi￾ent is prepared, the sample is layered on top, and the cen￾trifuge tubes are placed in the pockets of the vertical rotor. (b) Both sample and gradient begin to reorient as rotor accelerates. (c) Reori￾entation of the sample and gradient is now complete. (d) Bands form as the parti￾cles sediment. (e) Bands and gradient reorient as the rotor decelerates. (f) Bands and gradient both fully reori￾ented; rotor at rest. From Centifugation: A Practical Approach (2nd Ed.). 1984. Rickwood, D., ed. Reprinted by permission of Oxford Uni￾versity Press

he instrument is used, the more frequently these brushes need to be changed. This is a procedure that you can do yourself, since brushes ordered form the manufacturer usually provide detailed instructions. Most instruments are equipped with an indicator light hat signals a worn brush. As with any attempt to repair an instru ment, disconnect any power source and consult the manufacturer for warnings on any hazards. Cleanliness matters. Dirt and spilled materials can enter the motor compartment and cause failures. Clean any spills that occur inside the instrument as soon as possible When not in use, turn off the power to a refrigerated centrifuge nd open the chamber door to allow moisture to evaporate. If the instrument must be maintained with the power on, keep the nut as much as possible and check for frost before Check the level of an instrument after it has been moved. An unleveled instrument can cause damage to the drive mechanism a post-move preventative maintenance call can prevent problems machine is pe What Is the Best Way to Clean a Spill within a Centrifuge? Spills should be immediately cleaned using a manufacturer- approved detergent. Mild detergents are usually recommended and described in operating manuals. Avoid harsh solvents such as each and phenolIcs How Should You deal with a Walking Centrifuge? Older units are more prone to walking when imbalanced. If the instrument vibrates mildly, hit the stop button on the instrument If there is major shaking, cut the power to the instrument by a means other than the instruments power switch. You cant predict when or how the instrument is going to jump. An ultracentrifuge might require hours to come to a complete stop. Clear an area around the instrument and allow it to move if necessary. Vibra tion is going to be the greatest at a rotor-dependent critical speed usually below 2000rpm Never attempt to open the chamber door while the rotor is spinning, and don' t attempt to enter the rotor area even if the door is open. Don,t attempt to use physical force to restrict the movement of the machine. Keep your hands off the instrument until it comes to a complete stop How Can You Improve pellet Formation? Fluffy pellets that form on the side of a wall are easily dislodged during attempts to remove the supernate. To form tighter Troutman et al

the instrument is used, the more frequently these brushes need to be changed. This is a procedure that you can do yourself, since brushes ordered form the manufacturer usually provide detailed instructions. Most instruments are equipped with an indicator light that signals a worn brush. As with any attempt to repair an instru￾ment, disconnect any power source and consult the manufacturer for warnings on any hazards. Cleanliness matters. Dirt and spilled materials can enter the motor compartment and cause failures. Clean any spills that occur inside the instrument as soon as possible. When not in use, turn off the power to a refrigerated centrifuge, and open the chamber door to allow moisture to evaporate. If the instrument must be maintained with the power on, keep the chamber door shut as much as possible and check for frost before use. Check the level of an instrument after it has been moved. An unleveled instrument can cause damage to the drive mechanism. A post-move preventative maintenance call can prevent problems and ensure that the machine is performing accurately. What Is the Best Way to Clean a Spill within a Centrifuge? Spills should be immediately cleaned using a manufacturer￾approved detergent. Mild detergents are usually recommended and described in operating manuals. Avoid harsh solvents such as bleach and phenolics. How Should You Deal with a Walking Centrifuge? Older units are more prone to walking when imbalanced. If the instrument vibrates mildly, hit the stop button on the instrument. If there is major shaking, cut the power to the instrument by a means other than the instrument’s power switch.You can’t predict when or how the instrument is going to jump. An ultracentrifuge might require hours to come to a complete stop. Clear an area around the instrument and allow it to move if necessary. Vibra￾tion is going to be the greatest at a rotor-dependent critical speed, usually below 2000rpm. Never attempt to open the chamber door while the rotor is spinning, and don’t attempt to enter the rotor area even if the door is open. Don’t attempt to use physical force to restrict the movement of the machine. Keep your hands off the instrument until it comes to a complete stop. How Can You Improve Pellet Formation? Fluffy pellets that form on the side of a wall are easily dislodged during attempts to remove the supernate. To form tighter pellets, 66 Troutman et al

switch to a rotor with a steeper angle, or spin harder and longer at the existing angle. PIPETTORS (Michele A Kennedy) The accurate delivery of a solution is critical to almost all aspects of laboratory work. If the volume delivered is incorrect the results can be compounded throughout the entire experiment This section will discuss issues ranging from selecting the cor rect pipette from the start to ensuring that the pipette is working properly Which Pipette Is Most Appropriate for Your Application? Different applications will require the use of different types of pipettes or different methods of pipetting. Prior to purchasing a pipette, one should decide which type of pipette will be required to address the needs of the lab. There are two main types of pipettes: air displacement and positive displacement. The air dis- placement pipette is the most commonly used pipette in the lab In this type of pipette, a disposable pipette tip is used in conjunc tion with a pipette that has an internal piston. An air space, which is moved by the internal pipette piston, allows for the aspiration and dispensing of sample. This type of pipette is ideal for use with aqueous solutions. The second type of pipette is the positive displacement pipette In this pipette, the piston is contained within the disposable and comes in direct contact with the sample solution. Positive dis- placement pipettes are recommended for use with solutions that have a high vapor pressure or are very viscous. When pipettin solutions with a high vapor pressure, it is recommended to pre- wet the tip. This allows the small air space within a positive displacement system to become saturated with the vapors of the solution. Pre-wetting increases the accuracy of the pipetting because the sample will not evaporate into the saturated envi ronment, which would normally cause a pipette to leak. Once you have decided if an air or positive displacement pipette is the right choice for your lab, then the next thing to choose is the proper volume range. Determine what will be the most frequently used volume. This will then help you to decide on the style of pipette that will achieve the best accuracy. Fixed-volume pipettes provide the highest accuracy of manual pipettes, but they are limited to one volume. Adjustable-volume pipettes are slightly less accurate, but they allow for the pipetting of multiple volumes with one pipette. For example, an Eppendorf@ Series 2100 10 to 100ul How to Properly Use and Maintain Laboratory Equipment

switch to a rotor with a steeper angle, or spin harder and longer at the existing angle. PIPETTORS (Michele A. Kennedy) The accurate delivery of a solution is critical to almost all aspects of laboratory work. If the volume delivered is incorrect, the results can be compounded throughout the entire experiment. This section will discuss issues ranging from selecting the cor￾rect pipette from the start to ensuring that the pipette is working properly. Which Pipette Is Most Appropriate for Your Application? Different applications will require the use of different types of pipettes or different methods of pipetting. Prior to purchasing a pipette, one should decide which type of pipette will be required to address the needs of the lab. There are two main types of pipettes: air displacement and positive displacement. The air dis￾placement pipette is the most commonly used pipette in the lab. In this type of pipette, a disposable pipette tip is used in conjunc￾tion with a pipette that has an internal piston. An air space, which is moved by the internal pipette piston, allows for the aspiration and dispensing of sample. This type of pipette is ideal for use with aqueous solutions. The second type of pipette is the positive displacement pipette. In this pipette, the piston is contained within the disposable tip and comes in direct contact with the sample solution. Positive dis￾placement pipettes are recommended for use with solutions that have a high vapor pressure or are very viscous. When pipetting solutions with a high vapor pressure, it is recommended to pre￾wet the tip. This allows the small air space within a positive displacement system to become saturated with the vapors of the solution. Pre-wetting increases the accuracy of the pipetting because the sample will not evaporate into the saturated envi￾ronment, which would normally cause a pipette to leak. Once you have decided if an air or positive displacement pipette is the right choice for your lab, then the next thing to choose is the proper volume range. Determine what will be the most frequently used volume. This will then help you to decide on the style of pipette that will achieve the best accuracy. Fixed-volume pipettes provide the highest accuracy of manual pipettes, but they are limited to one volume.Adjustable-volume pipettes are slightly less accurate, but they allow for the pipetting of multiple volumes with one pipette. For example, an Eppendorf® Series 2100 10 to 100ml How to Properly Use and Maintain Laboratory Equipment 67

adjustable pi 100 ul has +O8%, whereas a Series 2100 100ul fixed-volume pipettes has an inaccuracy specification of +.6%. When choosing an adjustable volume pipette, remember that all adjustable pipettes pr greater accuracy at the high end of their volume range. An Eppen dorf Series 2100 10 to 100 ul adjustable pipette, set at 100 ul has an inaccuracy specification of +0.8%, whereas a 100 to 1000ul pipette set at 100 ul has an inaccuracy specification of +3.0% What Are the Elements of Proper Pipetting Technique? Once you have selected the correct pipette for your application then you must ensure that the pipette is used correctly. Improper use of a pipette can lead to variations in the volume being dis- pensed. When working with a pipette, check that all movements of the piston are smooth and not abrupt. Aspirating a sample too quickly can cause the sample to vortex, possibly overaspirating the a When aspirating a sample, it is important to make sure that the llowing guidelines are adhered to. First, the pipet only be immersed a few mm into the sampl spirated This ensures that the hydrostatic pressure is similar during aspi ration and dispensing. Next, the pipette should always be in a com- pletely vertical position during aspiration. The result of holding a pipette at an angle of 30 could create a maximum of +0.5% inaccuracy(Products and Applications for the Laboratory 2000, Eppendorf catalog, p. 161) When dispensing the sample, the pipette tip should touch the side of the receiving vessel. This will ensure the even flow of the sample from the tip, without forming droplets. If a droplet remains inside the tip, the volume dispensed will not be correct Preventing and solving Problems a good maintenance and calibration schedule is the ke ensuring that your pipette is working properly. More often than not,the factory-set calibration on a pipette is changed before a proper inspection and cleaning has been performed. A good maintenance program can prevent unnecessary changes in the calibration, which saves money over the life of the pipette A few maintenance suggestions are listed below: Always store pipettes in an upright position, preferably in a stand. This prevents the nose cones or pistons from being bent hen placed in a drawer Troutman et al

68 Troutman et al. adjustable pipette, set at 100ml has an inaccuracy specification of ±0.8%, whereas a Series 2100 100ml fixed-volume pipettes has an inaccuracy specification of ±0.6%. When choosing an adjustable￾volume pipette, remember that all adjustable pipettes provide greater accuracy at the high end of their volume range.An Eppen￾dorf Series 2100 10 to 100ml adjustable pipette, set at 100ml has an inaccuracy specification of ±0.8%, whereas a 100 to 1000ml pipette set at 100ml has an inaccuracy specification of ±3.0%. What Are the Elements of Proper Pipetting Technique? Once you have selected the correct pipette for your application, then you must ensure that the pipette is used correctly. Improper use of a pipette can lead to variations in the volume being dis￾pensed. When working with a pipette, check that all movements of the piston are smooth and not abrupt. Aspirating a sample too quickly can cause the sample to vortex, possibly overaspirating the sample. When aspirating a sample, it is important to make sure that the following guidelines are adhered to. First, the pipette tip should only be immersed a few mm into the sample to be aspirated (Figure 4.7). This ensures that the hydrostatic pressure is similar during aspi￾ration and dispensing. Next, the pipette should always be in a com￾pletely vertical position during aspiration. The result of holding a pipette at an angle of 30° could create a maximum of +0.5% inaccuracy (Products and Applications for the Laboratory 2000, Eppendorf® catalog, p. 161). When dispensing the sample, the pipette tip should touch the side of the receiving vessel. This will ensure the even flow of the sample from the tip, without forming droplets. If a droplet remains inside the tip, the volume dispensed will not be correct. Preventing and Solving Problems A good maintenance and calibration schedule is the key to ensuring that your pipette is working properly. More often than not, the factory-set calibration on a pipette is changed before a proper inspection and cleaning has been performed. A good maintenance program can prevent unnecessary changes in the calibration, which saves money over the life of the pipette. A few maintenance suggestions are listed below: • Always store pipettes in an upright position, preferably in a stand. This prevents the nose cones or pistons from being bent when placed in a drawer